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1.
J Immunol ; 206(11): 2652-2667, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34031145

RESUMO

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-κB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-κB recruitment in response to high LPS doses. Using the transposase-accessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections.


Assuntos
Cromatina/imunologia , Fatores de Transcrição/imunologia , Animais , Células Cultivadas , Desmetilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição/deficiência
2.
J Immunol ; 200(1): 305-315, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29150563

RESUMO

Macrophages are exquisite sensors of tissue homeostasis that can rapidly switch between pro- and anti-inflammatory or regulatory modes to respond to perturbations in their microenvironment. This functional plasticity involves a precise orchestration of gene expression patterns whose transcriptional regulators have not been fully characterized. We had previously identified the transcription factor NFAT5 as an activator of TLR-induced responses, and in this study we explore its contribution to macrophage functions in different polarization settings. We found that both in classically and alternatively polarized macrophages, NFAT5 enhanced functions associated with a proinflammatory profile such as bactericidal capacity and the ability to promote Th1 polarization over Th2 responses. In this regard, NFAT5 upregulated the Th1-stimulatory cytokine IL-12 in classically activated macrophages, whereas in alternatively polarized ones it enhanced the expression of the pro-Th1 mediators Fizz-1 and arginase 1, indicating that it could promote proinflammatory readiness by regulating independent genes in differently polarized macrophages. Finally, adoptive transfer assays in vivo revealed a reduced antitumor capacity in NFAT5-deficient macrophages against syngeneic Lewis lung carcinoma and ID8 ovarian carcinoma cells, a defect that in the ID8 model was associated with a reduced accumulation of effector CD8 T cells at the tumor site. Altogether, detailed analysis of the effect of NFAT5 in pro- and anti-inflammatory macrophages uncovered its ability to regulate distinct genes under both polarization modes and revealed its predominant role in promoting proinflammatory macrophage functions.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Neoplasias Experimentais/imunologia , Fatores de Transcrição/metabolismo , Animais , Arginase/metabolismo , Carcinoma Pulmonar de Lewis , Diferenciação Celular , Homeostase , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th1/imunologia , Células Th2/imunologia , Fatores de Transcrição/genética , Regulação para Cima
3.
Proc Natl Acad Sci U S A ; 110(40): 16091-6, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24043824

RESUMO

The Rel-like transcription factors nuclear factor kappa B (NF-κB) and the calcineurin-dependent nuclear factor of activated T cells (NFATc) control specific points of thymocyte maturation. Thymocytes also express a distinct member of the Rel family, the calcineurin-independent, osmostress response regulator NFAT5. Here we show that IKKß regulates the expression of NFAT5 in thymocytes, which in turn contributes to the survival of T-cell receptor αß thymocytes and the transition from the ß-selection checkpoint to the double-positive stage in an osmostress-independent manner. NFAT5-deficient thymocytes had normal expression and proximal signaling of the pre-T-cell receptor but exhibited a partial defect in ß-chain allelic exclusion and increased apoptosis. Further analysis showed that NFAT5 regulated the expression of the prosurvival factors A1 and Bcl2 and attenuated the proapoptotic p53/Noxa axis. These findings position NFAT5 as a target of the IKKß/NF-κB pathway in thymocytes and as a downstream effector of the prosurvival role of the pre-T-cell receptor.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/imunologia , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição NFATC/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais/imunologia , Timócitos/imunologia , Animais , Apoptose/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Transgênicos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Timócitos/citologia , Proteína Supressora de Tumor p53/metabolismo
4.
Front Biosci ; 13: 5359-73, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18508592

RESUMO

The human MAP kinase-interacting kinases (or MAP kinase signal-integrating kinases), Mnks, comprise a group of four proteins derived from two genes (Gene symbols: MKNK1 and MKNK2) by alternative splicing. Mnk1a/b differ at their C-termini, as do Mnk2a/2b: in each case, the a-form possesses a longer C-terminal region than the b-form, which lacks the MAP kinase-binding region. The N-termini of all forms contain a polybasic region which binds importin a and the translation factor scaffold protein eukaryotic initiation factor (eIF) 4G. The catalytic domains of Mnk1a/b and Mnk2a/b share three unusual features: two short inserts and a DFD feature where other kinases have DFG. Mnk isoforms differ markedly in their activity and regulation, and in subcellular localization. The best-characterised Mnk substrate is eIF4E. The cellular role of eIF4E phosphorylation remains unclear: it may promote export of certain mRNAs from the nucleus. Other Mnk substrates bind to AU-rich elements that modulate the stability/translation of specific mRNAs. Mnks may also control production of inflammatory mediators and signaling from tyrosine kinase receptors, as well as cell proliferation or survival.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Processamento Alternativo , Animais , Sobrevivência Celular , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Variação Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/enzimologia , Neoplasias/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética
5.
J Exp Med ; 215(11): 2901-2918, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30327417

RESUMO

MHCII in antigen-presenting cells (APCs) is a key regulator of adaptive immune responses. Expression of MHCII genes is controlled by the transcription coactivator CIITA, itself regulated through cell type-specific promoters. Here we show that the transcription factor NFAT5 is needed for expression of Ciita and MHCII in macrophages, but not in dendritic cells and other APCs. NFAT5-deficient macrophages showed defective activation of MHCII-dependent responses in CD4+ T lymphocytes and attenuated capacity to elicit graft rejection in vivo. Ultrasequencing analysis of NFAT5-immunoprecipitated chromatin uncovered an NFAT5-regulated region distally upstream of Ciita This region was required for CIITA and hence MHCII expression, exhibited NFAT5-dependent characteristics of active enhancers such as H3K27 acetylation marks, and required NFAT5 to interact with Ciita myeloid promoter I. Our results uncover an NFAT5-regulated mechanism that maintains CIITA and MHCII expression in macrophages and thus modulates their T lymphocyte priming capacity.


Assuntos
Elementos Facilitadores Genéticos/imunologia , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos/imunologia , Proteínas Nucleares/imunologia , Transativadores/imunologia , Fatores de Transcrição/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Rearranjo Gênico/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Macrófagos/citologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética
6.
Endocrinology ; 146(5): 2210-20, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15677760

RESUMO

The gene encoding the human TNF alpha receptor (TNFR) 2 contains polymorphisms in the 3' untranslated region (UTR). Previous studies have shown that some variant alleles in this region are associated with obesity and insulin resistance. However, the effect of these polymorphisms on the expression of TNFR2 has not been studied to date. To examine the role played by different haplotypes in the control of TNFR2 expression (haplotypes A1-A5, referring to nucleotides 1663 G/A, 1668 T/G, and 1690 T/C), we introduced these sequences into the 3'-UTR of a heterologous reporter gene and expressed the corresponding constructs in a human T-cell line. We demonstrate that a 485-nt fragment of the TNFR2 3'-UTR that contains a U-rich region decreases reporter expression and that haplotypes A1-A4 exert a stronger effect than A5. Furthermore, time-course assays of mRNA stability using actinomycin D revealed that haplotypes A1-A4 destabilize the mRNA. The proximal TNFR2 3'-UTR, independently of haplotype differences, responded to T-cell activation by increasing mRNA decay. Electromobility shift analysis demonstrated that protein(s) found in T-cell extracts bind to the 485-nt fragment. We suggest that an increased rate of TNFR2 mRNA decay protects cells from unrestrained TNF alpha effects and that this protection is weakened in A5 subjects. These findings may explain the association of this haplotype with obesity and increased leptin levels.


Assuntos
Regiões 3' não Traduzidas/genética , Regulação da Expressão Gênica , Genes Reporter/genética , Polimorfismo Genético/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Regiões 3' não Traduzidas/química , Regiões 3' não Traduzidas/metabolismo , Sequência de Bases , Citomegalovirus/genética , Dactinomicina/farmacologia , Estabilidade de Medicamentos , Citometria de Fluxo , Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Haplótipos , Humanos , Células Jurkat , Cinética , Leptina/sangue , Ativação Linfocitária , Dados de Sequência Molecular , Obesidade/genética , Regiões Promotoras Genéticas/genética , Estrutura Secundária de Proteína , RNA Mensageiro/análise , Receptores Tipo II do Fator de Necrose Tumoral/química , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Linfócitos T , Transfecção
7.
Sci Signal ; 7(332): re2, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24985347

RESUMO

The kinase mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation that integrates inputs from growth factor receptors, nutrient availability, intracellular ATP (adenosine 5'-triphosphate), and a variety of stressors. Since early works in the mid-1990s uncovered the role of mTOR in stimulating protein translation, this kinase has emerged as a rather multifaceted regulator of numerous processes. Whereas mTOR is generally activated by growth- and proliferation-stimulating signals, its activity can be reduced and even suppressed when cells are exposed to a variety of stress conditions. However, cells can also adapt to stress while maintaining their growth capacity and mTOR function. Despite knowledge accumulated on how stress represses mTOR, less is known about mTOR influencing stress responses. In this review, we discuss the capability of mTOR, in particular mTOR complex 1 (mTORC1), to activate stress-responsive transcription factors, and we outline open questions for future investigation.


Assuntos
Proliferação de Células/fisiologia , Biossíntese de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Fatores de Transcrição/metabolismo
8.
J Exp Med ; 209(2): 379-93, 2012 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-22312110

RESUMO

Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) ß activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.


Assuntos
Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes/imunologia , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Fatores de Transcrição/metabolismo , Animais , Imunoprecipitação da Cromatina , Primers do DNA/genética , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Quinase I-kappa B/metabolismo , Immunoblotting , Interleucina-6/metabolismo , Leishmania/imunologia , Luciferases , Camundongos , Camundongos Knockout , Análise em Microsséries , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Plasmídeos/genética , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/metabolismo
9.
J Biol Chem ; 283(1): 57-65, 2008 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-17965020

RESUMO

To identify new potential substrates for the MAP kinase signal-integrating kinases (Mnks), we employed a proteomic approach. The Mnks are targeted to the translational machinery through their interaction with the cap-binding initiation factor complex. We tested whether proteins retained on cap resin were substrates for the Mnks in vitro, and identified one such protein as PSF (the PTB (polypyrimidine tract-binding protein)-associated splicing factor). Mnks phosphorylate PSF at two sites in vitro, and our data show that PSF is an Mnk substrate in vivo. We also demonstrate that PSF, together with its partner, p54(nrb), binds RNAs that contain AU-rich elements (AREs), such as those for proinflammatory cytokines (e.g. tumor necrosis factor alpha (TNFalpha)). Indeed, PSF associates specifically with the TNFalpha mRNA in living cells. PSF is phosphorylated at two sites by the Mnks. Our data show that Mnk-mediated phosphorylation increases the binding of PSF to the TNFalpha mRNA in living cells. These findings identify a novel Mnk substrate. They also suggest that the Mnk-catalyzed phosphorylation of PSF may regulate the fate of specific mRNAs by modulating their binding to PSF.p54(nrb).


Assuntos
Proteínas Associadas à Matriz Nuclear/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Necrose Tumoral alfa/genética , Regiões 3' não Traduzidas/metabolismo , Linhagem Celular , Cromatografia de Afinidade , Proteínas de Ligação a DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Immunoblotting , Imunoprecipitação , Células Jurkat , Proteínas Associadas à Matriz Nuclear/genética , Fatores de Transcrição de Octâmero/genética , Fator de Processamento Associado a PTB , Fosforilação , Ligação Proteica , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Transfecção
10.
J Biol Chem ; 280(45): 37623-33, 2005 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-16162500

RESUMO

The MAPK signal-integrating kinases Mnk1 and Mnk2 are closely related but show marked differences in their basal activities and regulation. Both possess, within their C termini, motifs for binding to MAPKs, although these differ between Mnk1 and Mnk2. Mnk2 shows much higher activity in unstimulated cells than Mnk1, whose activity is greatly increased, e.g. by stimulation of the MEK/ERK pathway. Such increases are sensitive to blockade of that pathway, whereas the activation state of Mnk2 is relatively insensitive to inhibition of upstream signaling. Here we have studied the roles of features in their catalytic domains and C termini in determining their regulatory properties and basal activities. Mnk2 can bind to phosphorylated, active ERK, whereas Mnk1 cannot. Such binding apparently protects ERK against dephosphorylation and inactivation. The high basal activity of Mnk2 and its binding to (phospho)ERK requires features both of the catalytic domain and of the C terminus. For example, within the catalytic region an aspartate in Mnk2 plays a key role. Mutation to alanine inactivates Mnk2. In the C terminus, features within the MAPK-binding motif and to either side of it, including potential phosphorylation sites, affect MAPK binding and activity. The association of Mnks with the scaffold protein eukaryotic initiation factor 4G is negatively modulated by Mnk activity. These data indicate that multiple features determine the activities of the Mnks and thus impact on their ability to phosphorylate physiological substrates such as eukaryotic initiation factor 4E.


Assuntos
Domínio Catalítico , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Fator de Iniciação Eucariótico 4G/metabolismo , Flavonoides/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Homologia de Sequência de Aminoácidos
11.
Immunity ; 23(2): 177-89, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16111636

RESUMO

Posttranscriptional regulatory mechanisms control TNFalpha expression through AU-rich elements in the 3'UTR of its mRNA. This is mediated through Erk and p38 MAP kinase signaling, although the mechanisms involved remain poorly understood. Here, we show that the MAP kinase signal-integrating kinases (Mnks), which are activated by both these pathways, regulate TNFalpha expression in T cells via the 3'UTR. A selective Mnk inhibitor or siRNA-mediated knockdown of Mnk1 inhibits TNFalpha production in T cells, whereas Mnk1 overexpression enhances expression of a reporter construct containing the TNFalpha 3'UTR. We identify ARE binding proteins that are Mnk substrates, such as hnRNP A1, which they phosphorylate at two sites in vitro. hnRNP A1 is phosphorylated in response to T cell activation, and this is blocked by Mnk inhibition. Moreover, Mnk-mediated phosphorylation decreases binding of hnRNP A1 to TNFalpha-ARE in vitro or TNFalpha-mRNA in vivo. Therefore, Mnks are novel players in cytokine regulation and potential new targets for anti-inflammatory therapy.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Regiões 3' não Traduzidas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Reporter , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/antagonistas & inibidores , Humanos , Células Jurkat , Fosforilação , Ligação Proteica/genética , Ligação Proteica/imunologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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